Methods of monitoring HIV drug resistance

ABSTRACT

A method is provided for detecting a presence of HIV virus in a sample comprising: taking a culture of recombinant cells which (a) are capable of cell division, (b) express CD4 receptor and one or more additional cell surface receptors necessary to allow the HIV virus to infect, (c) enable the HIV virus to replicate and infect the noninfected cells in the cell culture, and (d) comprise a reporter sequence introduced into the recombinant cells comprising a reporter gene whose expression is regulated by a protein specific to HIV viruses which is expressed from a genome of an HIV virus upon infection of the recombinant cell by the HIV virus; contacting the cell culture with a sample to be analyzed for the presence of HIV virus in the sample; and detecting a change in a level of expression of the reporter gene in cells in the recombinant cell culture. The method can be used to detect the presence of HIV virus in a sample, detect the presence of different strains of HIV virus in a sample, detect HIV drug resistance in a sample, determine what combination of one or more anti-HIV agents would be effective in treating a patient, and screen compositions for anti-HIV activity.

RELATIONSHIP TO COPENDING APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/314,259, filed May 18, 1999, now U.S. Pat. No. 6,406,911, claimsbenefit of U.S. Provisional Application No. 60/117,136, filed Jan. 25,1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to recombinant cell lines and methods fordetecting and monitoring viral infection. More particularly, theinvention relates to recombinant cell lines and methods for detectingHIV infection, monitoring HIV for drug resistance and screening foranti-HIV agents.

2. Description of Related Art

Human immunodeficiency virus (HIV) has been implicated as the primarycause of the slowly degenerate disease of the immune system termedacquired immune deficiency syndrome (AIDS). Infection of the CD4⁺subclass of T-lymphocytes with the HIV type-1 virus (HIV-1) leads todepletion of this essential lymphocyte subclass which inevitably leadsto opportunistic infections, neurological disease, neoplastic growth andeventual death.

Infection with human immunodeficiency virus (HIV) is a chronic processwith persistent, high rates of viral replication. The pathogenesis ofHIV-1 infection is characterized by a variable but often prolongedasymptomic period following the acute viremic phase. Previous work hasestablished a correlation between HIV disease progression and increasingamounts of infectious virus, viral antigens, and virus-specific nucleicacids (Ho et al., New England. J. Med. 321: 1621-1625 (1989); Schnittmanet al. AIDS Res. Hum. Retroviruses 7: 361-367 (1991); Pantalco et al.Nature 362: 355-358 (1993)).

A variety of reagents and assays have been developed to detect theinfection of HIV and monitor the progression of HIV in the body. Forexample, counting the depletion of CD4+ cells has been used to indicatethe prognosis of AIDS. Serological screening techniques are also beingutilized worldwide for the detection of HIV, where the presence of theantibody against HIV antigens, such as the HIV p24 antigen, is detected.

An ELISA assay is currently being utilized on serum samples in mosthospitals and screening laboratories to make the determination. However,currently used ELISA assays may not be sensitive enough to detect allHIV infected individuals. This is because that some HIV infectedindividuals do not have detectable levels of serum antibody to HIV.There may be a significant time lag between detection of HIV infectionand seroconversion. In addition, some HIV infected but seronegativeindividuals might never convert but will remain infected throughouttheirs lives. Thus, such a screening method may generate falsenegatives, which in turn may increases the probability of HIV infectionof healthy people by these individuals.

Another method for detecting HIV infection in seronegative individualswas described (Jehuda-Cohen, T. et al. Proc. Natl. Acad. Sci. UAS, 87:3972-3076 (1990)) wherein peripheral blood mononuclear cells (PBMC) areisolated from the blood and then exposed to a mitogen such as pokeweedmitogen. Incubation of isolated PBMC with pokeweed mitogen caused thePBMC to secret immunoglobulins that were specific for HIV. The failureof the ELISA assay to detect all HIV infected individuals places thepopulation at risk by misleading the HIV infected individuals that theyare not infected, thereby making it more likely that the HIV infectedindividuals will unknowingly infect others.

The existence of HIV has also been determined by using the reversetranscriptase-polymerase chain reaction (RT-PCR) to amplify plasma HIVRNAs (U.S. Pat. No. 5,674,680). This method is used to detect threetypes of HIV mRNA in peripheral blood cells: unspliced, multiplespliced, and single-spliced mRNA in AIDS patients, HIV-infected butasymptomatic individuals and individuals who are undergoing therapy forAIDS. However, the correlation between the differences in HIV mRNAlevels and AIDS prognosis needs to be established.

Many antiviral drugs have been developed to inhibit HIV infection andreplication by targeting HIV reverse transcriptase and proteases.Treatment following a prolonged single drug regimen has met with limitedsuccess where there is relatively small drop in viral load, followed bya rise in amount of detectable virus in blood, presumably due to thedevelopment of drug resistance strains of HIV. The resistance of HIV todrugs is not only associated with the high mutation rates of HIV butalso due to the selective pressure of prolonged anti-HIV drug therapy.Since the original description of diminished susceptibility of isolatesof HIV-1 to zidovudine (AZT) (Larder et al. Science (1989)243:1731-1734), the literature has disclosed many descriptions ofdiminished susceptibility to AZT in different clinical situations, withdifferent assay systems, and of genetic mutations responsible forchanges in susceptibility. For example, isolates from subjects nottreated with AZT display a narrow range of susceptibilities to AZT, withthe 50% inhibitory concentrations (IC50) ranging from 0.001 to 0.04 μM(Larder et al. (1989), supra; Rooke et al. AIDS (1989) 3:411-415; Landet al. J Infect Dis (1990) 161:326-329; Richman et al. J. AIDS (1990)3:743-746; Tudor-Williams et al. Lancet (1992) 339:15-19). This narrowrange of susceptibilities is typical for HIV isolates from subjects ofall ages and at all stages of HIV infection. Isolates of HIV frompatients who receive AZT, however, chronically display progressivereductions of susceptibility to AZT over periods of months to years.Diminished susceptibility to AZT of an isolate of HIV-2 from a patienton prolonged therapy has also been reported (Pepin et al. EighthInternational Conference on AIDS, Amsterdam, The Netherlands, Jul.19-24, 1992 Abstract PoA 24401).

In addition to AZT, HIV resistance have been seen with other nucleosidesand to nonnucleoside anti-retroviral drugs. For example, isolatesresistant to AZT display diminished susceptibility to other nucleosidescontaining a 3′-azido moiety, including 3′-azido-2′,3′-dideoxyuridine,3′-azido-2′,′dideoxyguanosine, and 3′-azido-2′,3′-dideoxyadenosine(Larder et al. (1989), supra; Larder et al. Antimicrob Agents Chemother(1990) 34:436-441). Additionally, AZT-resistant isolates are reported todisplay cross-resistance to didehydrodideoxythymidine (Rooke et al.Antimicrob. Agents Chemother. (1991) 35:988-991).

Drug resistance in HIV isolates is not limited to inhibitors of reversetranscriptase and virtually all drug targets for anti-HIV therapy aresusceptible to the development of resistance. For example, a mutant withresistance to a protease inhibitor has been isolated that exhibits aneightfold reduction in susceptibility to a protease inhibitor (Pattersonet al. Eighth International Conference on AIDS, Amsterdam, TheNetherlands, Jul. 19-24, 1992, Abstract ThA 1506).

In the last five year, with the fast development of anti-HIV drugs andutilization of combination therapy, treatment of HIV infection withmultiple antiviral drugs (“cocktails”) have led to diminutions in theamount of viral RNA and virus detectable in blood by using currentdetection methods. It has been shown that combination therapy with 3 ormore antiviral drugs, e.g. indinavir, zidovudine, and lamivudine, oralternatively, nevirapine, zidovudine, and didanosine, in previouslyuntreated patients has resulted in profound decreases in viral burden(Wainberg, M. A. and Friedland, G. JAMA (1998) 279: 1977-1983). It wasbelieved that the combination antiviral regimens used must have blockedviral replication to the extent that the mutations that encode drugresistance could not occur. However, current studies showed that agrowing number of patients are failing combination drug regimens (Deek,S. et al. the 5th Conference on Retroviruses and OpportunisticInfection, Chicago, Feb. 1-5, 1998, Abstract #419). Finding an effectivesalvage therapy for them is difficult.

In the clinical setting, drug resistance is often not detected until apatient manifests symptoms of disease progression, which is generallynot observed until significantly after development of a drug resistantstrain of virus. Thus, there is a clear need for an assay which canindicate the drug resistance of virus strains so drug therapy for apatient can be modified accordingly, and optimally as soon as resistanceis detected rather than delaying until clinical symptoms are observed.

Currently the most commonly used assays for susceptibility of HIV toantiviral drugs involve the measurement of the inhibition ofcytopathology, p24 production, or reverse transcriptase production of alaboratory strain of HIV in a lymphoblastoid cell line. Such assays maynot be readily applied to clinical isolates of HIV. Examples of commonlyused assays of drug susceptibility of clinical isolates have been thesyncytial focus assay in CD4-HeLa cells (Chesebro, B. and Wehrly, K., J.Virol. (1988) 62:3779-3788), inhibition of p24 production in primaryperipheral blood mononuclear cells, and reverse transcriptase (RT)assays using cultured primary T-cells from patient blood. (Richman etal. In: Current Protocols in Immunology, Coligan et al., eds, (1993)Brooklyn, J. Wiley).

One of the disadvantages associated with the syncytial focus assay isthat it may only detect HIV viruses that exhibit a syncytial-inducingphenotype and that in practice may only be obtained from a minority ofspecimens from seropositive individuals. And the syncytial focus assaysmay not be used for screening for drugs that affect posttranslationalprocessing, such as glycosidase and protease inhibitors. On the otherhand, the p24 and RT assays may also suffer the limitations of difficultquantitation, low sensitivity and unproven clinical validity.

SUMMARY OF THE INVENTION

A recombinant cell is provided which comprises: a reporter sequenceintroduced into the recombinant cell comprising a reporter gene whoseexpression is regulated by a protein specific to HIV viruses which isexpressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; the recombinant cell being capable ofcell division and expressing a CD4 receptor and one or more additionalcell surface receptors which facilitate productive infection of therecombinant cell by the HIV virus; and the recombinant cell enabling HIVvirus which has infected the recombinant cell to replicate and infectnon-infected cells in a culture of the recombinant cell.

As used herein, introducing a reporter sequence into a recombinant cellrefers to the introduction of a sequence into cell by any of a varietyof recombinant methodologies including, but not limited to,transformation, transfection and transduction.

The recombinant cell may optionally express a sufficient number of cellsurface receptors to render the recombinant cell permissive tosubstantially all strains of HIV. Alternatively, the recombinant cellmay express a selected group of cell surface receptors such that therecombinant cell is permissive to a selected group of strains of HIV.Examples of cell surface receptors which may be expressed by therecombinant cell include, but are not limited to CXCR4, CCR5, CCR1,CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO andGPR15/BOB.

The stably transferred reporter sequence may optionally comprise apromoter sequence including an HIV virus specific enhancer sequence, anda reporter gene whose expression is regulated by binding of an HIVspecific transactivator protein to the HIV specific enhancer sequence.According to this variation, the HIV specific transactivator protein ispreferably Tat and the HIV specific enhancer sequence preferablycomprises at least one copy of TAR sequence. Alternatively, the HIVspecific protein may optionally regulates expression of the reportersequence by a protein-protein interaction between the HIV specificprotein and a transactivator protein present in the recombinant cell.

Examples of the HIV specific protein include, but are not limited to,HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef, Gag, Env, RT, PR, andIN. The HIV specific protein may optionally be an HIV transactivatorprotein such as Tat.

Expression of the reporter gene in the recombinant cell may be isup-regulated or down-regulated by the HIV specific protein.

A method is provided for detecting a presence of HIV virus in a samplecomprising: taking a culture of recombinant cells which (a) are capableof cell division, (b) express CD4 receptor and one or more additionalcell surface receptors necessary to allow the HIV virus to infect, (c)enable the HIV virus to replicate and infect the noninfected cells inthe cell culture, and (d) comprise a reporter sequence introduced intothe recombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell culture with a sample to be analyzed for thepresence of HIV virus in the sample; and detecting a change in a levelof expression of the reporter gene in cells in the recombinant cellculture.

A method is also provided for detecting the presence of differentstrains of HIV virus in a sample comprising: taking a first culture ofrecombinant cells which (a) are capable of cell division, (b) expressCD4 receptor and one or more additional cell surface receptors whichrender the first cell culture permissive to a first group of strains ofHIV but does not render the first cell culture permissive to a second,different group of strains of HIV, (c) enable the HIV virus to replicateand infect the noninfected cells in the cell culture, and (d) comprise areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus; taking a second culture ofrecombinant cells which (a) are capable of cell division, (b) expressCD4 receptor and one or more additional cell surface receptors whichrender the second culture permissive to the second group of strains ofHIV but does not render the second cell culture permissive to the firstgroup of strains of HIV, (c) enable the HIV virus to replicate andinfect the noninfected cells in the cell culture, and (d) comprise areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus; contacting the first andsecond cell cultures with a sample to be analyzed for the presence ofdifferent strains of HIV virus; detecting a change in a level ofexpression of the reporter gene in cells in the first cell culture;detecting a change in a level of expression of the reporter gene incells in the second cell culture; and distinguishing between the firstand second groups of strains based on whether a change in a level ofexpression of the reporter gene occurs in the first or the second cellculture.

According to the above method, the first and second cultures ofrecombinant cells may optionally be mixed with each other. The reportergenes in the first and second cultures of recombinant cells may alsooptionally be different from each other so that cells of the first cellculture can be distinguished from cells of the second cell culture. Thisallows different strains of HIV virus to be detected in a single wellcontaining cells from both cultures.

A method is also provided for detecting HIV drug resistance in a samplecomprising: taking a culture of recombinant cells which (a) are capableof cell division, (b) express CD4 receptor and one or more additionalcell surface receptors necessary to allow the HIV virus to infect, (c)enable the HIV virus to replicate and infect the noninfected cells inthe cell culture, and (d) comprise a reporter sequence introduced intothe recombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell culture with a sample containing HIV virus;adding one or more anti-HIV agents to the cell culture either before orafter contacting the cell culture with the sample; and detecting achange in a level of expression of the reporter gene in the cells.

A method is also provided for taking a patient known to be infected withone or more strains of the HIV virus and determining what combination ofone or more anti-HIV agents would be effective in treating the patient,the method comprising: taking a plurality of cell cultures, each of thecultures containing recombinant cells which (a) are capable of celldivision, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in the cellculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell cultures with a sample containing the HIVvirus; adding a different set of one or more anti-HIV agents to each ofthe cell cultures, either before or after contacting the cell cultureswith the sample; and comparing expression of the reporter gene in theplurality of cell cultures.

A method for screening compositions for anti-HIV activity comprising:taking a culture of recombinant cells which (a) are capable of celldivision, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in the cellculture, and (d) comprise a reporter sequence introduced, into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus; contacting the cell culture with a sample containing the HIVvirus; adding one or more agents whose anti-HIV activity are unknown tothe cell culture, either before or after contacting the cell cultureswith the sample; and detecting a change in a level of expression of thereporter gene in the cells in the culture.

According to any one of the above methods, the recombinant cells in thecell cultures used in the methods may optionally comprise a reportersequence introduced into the recombinant cells comprising a reportergene whose expression is regulated by a protein specific to HIV viruseswhich is expressed from a genome of an HIV virus upon infection of therecombinant cell by the HIV virus; the recombinant cells being capableof cell division and expressing a CD4 receptor and one or moreadditional cell surface receptors which facilitate productive infectionof the recombinant cell by the HIV virus; and the recombinant cellsenabling the HIV virus which has infected the recombinant cell toreplicate and infect non-infected cells in a culture of the recombinantcell.

Also according to any one of the above methods, the HIV specific proteinmay be any one of the HIV proteins Tat, Rev, Vpr, Vpx, Vif, Vpu, Nef,Gag, Env, RT, PR, and IN. The HIV specific protein may optionally be anHIV transactivator protein such as Tat.

Also according to any one of the above methods, the reporter sequencemay comprise a promoter sequence including an HIV virus specificenhancer sequence, and a reporter gene whose expression is regulated bybinding of an HIV specific transactivator protein to the HIV specificenhancer sequence. In one variation, the HIV specific transactivatorprotein is Tat and the HIV specific enhancer sequence comprises at leastone copy of TAR sequence.

Also according to any one of the above methods, the one or moreadditional cell surface receptors expressed by the recombinant cell mayinclude, but are not limited to CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4,CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO and GPR15/BOB.

Also according to any one of the above methods, detecting a change in alevel of expression of the reporter gene in the cells may includedetecting a change in a level of expression of the reporter gene inindividual cells.

Also according to any one of the above methods, detecting a change in alevel of expression of the reporter gene in the cells may includedetecting a change in a level of expression of the reporter gene acrossthe cell culture.

Also according to any one of the above methods, detecting a change in alevel of expression of the reporter gene in the cells may includedetecting whether viral replication within the cell culture hasoccurred.

Also according to any one of the above methods, detecting a change in alevel of expression of the reporter gene in the cells may includecomparing a level of expression in cells contacted with the sample to alevel of expression cells contacted with one or more control samples.

Also according to any one of the above methods, the sample may be anysample which might include HIV including, but not limited to wholeblood, blood serum, isolated peripheral blood cells,T cells, and bonemarrow.

Kits are also provided for performing the various methods of the presentinvention. These kits may include the cell line of the present inventionand any two or more components used to perform these methods.

In one variation, a kit is provided which comprises: first and secondrecombinant cell lines, each recombinant cell line comprising: areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus, the recombinant cell linebeing capable of cell division and expressing a CD4 receptor and one ormore additional cell surface receptors which facilitate productiveinfection of the recombinant cell by the HIV virus, and the recombinantcell line enabling the HIV virus which has infected the recombinant cellto replicate and infect non-infected cells in a culture of therecombinant cell; wherein the one or more additional cell surfacereceptors which the first recombinant cell line expresses renders thefirst recombinant cell line permissive to a first group of strains ofHIV and the one or more additional cell surface receptors which thesecond recombinant cell line expresses renders the second recombinantcell line permissive to a second, different group of strains of HIV.

According to this variation, the first and second recombinant cell linesmay optionally be mixed together in the kit. Also according to thisvariation, the first recombinant cell line may optionally include afirst reporter gene and the second recombinant cell line may optionallyinclude a second different reporter gene which allows the first andsecond recombinant cell lines to be independently identified.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates expression plasmids for HIV receptors and a reportergene.

FIG. 1B illustrates retroviral vectors for HIV receptors and a reportergene.

FIG. 2A illustrates an expression plasmid for human CD4 and CXCR4receptors.

FIG. 2B illustrates a plasmid for a lacZ reporter gene.

FIG. 3A shows HeLaT4 cells cultured in the presence of HIV virus andlater processed with X-Gal.

FIG. 3B shows HeLa D4R4 cells cultured in the presence of HIV virus andlater processed with X-Gal 1 day after the initial infection.

FIG. 3C shows HeLa D4R4 cells cultured in the presence of HIV virus andlater processed with X-Gal 3 days after the initial infection.

FIG. 3D shows HeLa D4R4 cells cultured in the presence of HIV virus andlater processed with X-Gal 4 days after the initial infection.

FIG. 3E shows HeLa D4R4 cells cultured in the presence of HIV virus andlater processed with X-Gal 5 days after the initial infection.

FIG. 3F shows HeLa D4R4 cells cultured in the presence of HIV virus andAZT and later processed with X-Gal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new and useful methods includingmethods for detecting HIV, methods for detecting HIV drug resistance,methods for designing patient customized anti-HIV drug cocktailtreatments, and methods for screening compositions for anti-HIVactivity. Also provided are novel cell lines which may be used with themethods of the present invention.

The methods of the present invention use cells which (a) are capable ofcell division; (b) are permissive to HIV virus; (c) express a reportergene whose expression is selectively regulated by infection with HIV;and (d) allow viral replication of HIV in infected cells which enablescells within the same cell culture which are initially uninfected tobecome infected.

One of the advantages provided by the present invention is that therecombinant cells used are capable of cell division. As a result, it iseasy to produce and maintain these cells for performing the variousmethods of the present invention.

A further advantage provided by the present invention is that therecombinant cells can be infected by multiple different strains of HIV,including wild-type and mutant HIV strains from clinical isolates orlaboratory-adapted strains. As a result, the methods of the presentinvention have broad applicability to all strains of HIV.

Yet a further advantage provided by the present invention is thatinfection of the recombinant cells by an HIV virus can be easilymonitored and measured. By using a reporter gene whose expression isregulated by infection with HIV, it is possible to detect HIV infectionby simple detection methods, such as calorimetric methods. By expressionof the reporter gene being selectively regulated by infection with HIV,false positive signals, for example due to infection by non-HIV viruses,are reduced.

A further advantage of the present invention is that the recombinantcells not only allow entry and infection of the HIV virus, but alsofacilitate efficient replication within the recombinant cell andtransmission of the mature HIV virion to infect other cells in theculture. By using a cell line in which HIV is able to infect some cellsin a cell culture, replicate, and then infect other cells in the cellculture, as well as by coupling viral replication with cell division,the signal produced by the reporter gene is amplified since more cellsare infected than would be infected absent replication of HIV within thecell culture. For example, a single virion contained in a sample isultimately able to infect all cells in the cell culture. This featureallows for sensitive detection of the HIV virus contained in a samplethat is applied to the recombinant cell culture.

By exploiting the above-described advantages, as well as featuresfurther described in details below, the recombinant cell line can beused in a variety of methods or assays for many laboratory and clinicalapplications relating to HIV.

It should be noted that the methods and cells of the present inventioncan be modified and adapted for various viruses other than HIV,including but are not limited to retroviruses, coronaviruses, herpesviruses and adenoviruses. For example, an immortalized cell line can beconstructed to comprise a panel of receptors and coreceptors to allowinfection, replication and amplification of one or more strains of atarget virus; and a reporter gene whose expression is regulated by aspecific gene product expressed by the target virus.

1. Recombinant Cell Line

One aspect of the present invention relates to recombinant cells for usein detecting infection by an HIV virus. In one embodiment, therecombinant cell comprises:

a reporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus;

the recombinant cell being capable of cell division and expressing a CD4receptor and one or more additional cell surface receptors whichfacilitate productive infection of the recombinant cell by the HIVvirus; and

the recombinant cell enabling the HIV virus which has infected therecombinant cell to replicate and infect non-infected cells in a cultureof the recombinant cell.

Regulation of the reporter gene expression may involve up-regulationwhere the HIV specific protein causes expression of the reporter gene tobegin or to increase. Alternatively, regulation of the reporter geneexpression may involve down-regulation where the HIV specific proteincauses expression of the reporter gene to cease or to decrease.

The HIV specific protein may be an HIV transactivator proteins such asTat, an HIV regulatory protein such as Rev, HIV accessory proteins suchas Vpr, Vpx, Vif, Vpu and Nef, HIV structural proteins such as Gag andEnv, or HIV enzymatic proteins such as RT (reverse transcriptase), PR(protease) and IN (integrase). The regulation of the reporter sequencemay be achieved by using various methods known in the art. For exampleexpression of the reporter sequence can be regulated by direct bindingof the transactivator protein Tat to an enhancer sequence upstreamcomprising at least one copy of TAR sequence. Alternatively, expressionof the reporter gene can be regulated via protein-protein interactionbetween the HIV specific protein and an transactivator protein presentin the recombinant cell.

In one variation of this embodiment, the reporter sequence in therecombinant cell comprises a promoter sequence including an HIV virusspecific enhancer sequence, and a reporter gene whose expression isregulated by binding of an HIV specific transactivator protein to theHIV specific enhancer sequence.

According to this preferred embodiment, regulation of the reporter geneexpression in the recombinant cells is achieved by using a promotersequence including an HIV virus specific enhancer sequence which istranscriptionally responsive to an HIV specific transactivator protein.Upon infection by the HIV virus, the HIV specific transactivator proteinexpressed from the HIV genome binds to the HIV specific enhancersequence and enhances expression of the reporter gene. The presence,absence or level of the reporter gene product is detected and used toindicate the infection of the HIV virus.

In a particularly preferred variation, the reporter sequence comprisesat least one copy of TAR sequence as the HIV virus specific enhancersequence. Expression of the reporter sequence is regulated by thebinding of the HIV specific transactivator protein Tat to the enhancersequence TAR.

a wide variety of reporter genes may be used in the present invention,Examples of proteins encoded by reporter genes include, but are notlimited to, easily assayed enzymes such as β-galactosidase, luciferase,beta-glucuronidase, chloramphenicol acetyl transferase (CAT), secretedembryonic alkaline phosphatase (SEAP), fluorescent proteins such asgreen fluorescent protein (GFP), enhanced blue fluorescent protein(EBFP), enhanced yellow fluorescent protein (EYFP) and enhanced cyanfluorescent protein (ECFP); and proteins for which immunoassays arereadily available such as hormones and cytokines. The expression ofthese reporter genes can also be monitored by measuring levels of mRNAtranscribed from these genes.

The one or more additional cell surface receptors expressed by therecombinant cell may optionally include, but are not limited to, CXCR4,CCR5, other chemokine receptors such as CCR1, CCR2b, CCR3, CCR4, CCR8,CXCR1, CXCR2, CXCR3, CX₃CR1, and chemokine receptor-like orphan proteinssuch as STRL33/BONZO and GPR15/BOB.

The presence of CD4 and these one or more additional cell surfacereceptors allows efficient entry, infection and replication of HIVstrains with different tropisms. By causing the recombinant cell toexpress as many cell surface receptors as possible, the recombinant cellmay be rendered permissive to virtually all strains of HIV, regardlessof tropism. This may be accomplished by transfecting or transducing thecell with all cell surface receptors known to be involved in HIVinfection or by cell fusion with cells, such as T-cells or monocytes,which express these receptors on the cell surface. Alternatively, bycausing the recombinant cell to express certain cell surface receptorsor sets of cell surface receptors, it is possible to design therecombinant cell to be permissive to certain strains of HIV and to notbe permissive to other strains of HIV. Thus, by selecting which cellsurface receptors are expressed, cell lines can be designed forscreening for particular strains or groups of strains of HIV virus.

The recombinant cell lines used in the present invention can beconstructed from a wide variety of immortalized cell lines. In oneembodiment, the recombinant cells are immortalized tumor cells. One ofthe advantages associated with using tumor cells is that tumor cellsundergo relatively fast cell cycling or division, which may furtherenhance replication and amplification of the virus in the culture. Theimmortalized tumor cell lines can be generated from primary tumor cellsor from established tumor cell lines. Alternatively, normal cells canalso be used so long as the cells are immortalized. Examples include butare not limited to primary cells immortalized by transfection withtelomerase gene and normal cells immortalized by SV40 tranformation.These immortalized cells can proliferate indefinitely, thus providing anample and economic supply of cells.

Compared to human T-cells that have been used in the art for HIV virusproduction, the recombinant cell lines of the present invention arerelatively easier to culture, more stable, and less expensive. It hasbeen acknowledged that the principle cell types targeted by HIV-1 arehelper T-lymphocytes and cells of the monocyte macrophage lineage viathe CD4 receptor pathway in vivo, while in tissue culture systems, HIVare cytopathic for CD4⁺-lymphocytes and cause dysfunction ofmacrophages, which is directly accounted for depletion of T cells in thebody. Since replicating HIV in infected individuals is readily detectedin peripheral blood and lymph lodes, human peripheral mononuclear cells(PBMC), in particular, have been frequently used as host cells for HIVinfection in vitro and anti-HIV drug-susceptibility testing. One of thedisadvantages with PBMC cells is that these primary cells have to beobtained from donors, carefully cultured and freshly prepared each time.It is costly and inefficient to use these primary T-cells for commercialpurposes. In addition, the permissiveness of these T-cells to differentstrains of HIV virus may vary with the donor, thus causing ambiguity inclinical testing. Thus, the recombinant cells of the present inventionwhich can be produced in an ample supply, are permissive to HIVinfection, relatively stable and can be cultured and manipulated moreeasily in vitro, are well suited for large scale commercial reproductionand use in high throughput screening.

2. Methods for Detecting HIV in a Sample

Methods are provided for detecting a presence of HIV virus in a sample.In one embodiment, the method comprises:

taking a culture of recombinant cells, which (a) are capable of celldivision, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

contacting the cell culture with a sample to be analyzed for thepresence of HIV virus in the sample; and

detecting a change in a level of expression of the reporter gene incells in the culture, such change being indicative of the HIV virusbeing present in the sample and infecting cells in the cell culture.

The culture of recombinant cells used in the method may be any cellculture which has the above described properties. The recombinant cellsdescribed in Section I are examples of cells having these properties andmay be used in this method.

Detecting a change in a level of expression of the reporter gene in thecells in the culture may be performed by detecting a change in a levelof expression of the reporter gene in individual cells or a change in alevel of expression of the reporter gene across the cell culture.

In one embodiment, detecting a change in a level of expression includesdetecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

In another embodiment, detecting a change in a level of expressionincludes comparing a level of expression in cells contacted with thesample to a level of expression cells contacted with one or more controlsamples. For example, cells contacted with a sample not containing HIVvirus can serve as a negative control, while cells contacted with asample containing HIV virus, recombinant and stabilized HIV virus, oranother virus capable of infecting the cells and causing expression ofthe HIV specific protein, such as a modified adenovirus encoding Tat,can serve as a positive control. By using suitable controls, inductionof the reporter gene expression may be better correlated with HIVinfection.

It is noted that regulation of the reporter gene may be up regulation ordown regulation. Accordingly, a change in the level of expression of thereporter gene may be an increase or decrease in reporter geneexpression.

The methods described above can be used for diagnosis of HIV viruscontained in variety of samples including, but are not limited to, wholeblood, blood serum, isolated peripheral blood cells, T cells, otherbiological fluids such as urine, saliva, tears and semen, as well asisolated wild-type or mutant HIV viruses from laboratories or clinics.For example, whole blood of individuals can be tested for the presenceof HIV virus by using the methods described above. In addition, blood orbone marrow samples from individual donors or samples from pooled bloodstored in blood banks can be screened for the presence of HIV virus. Thesensitivity of the methods to detect even a single HIV virion allows forthe diagnosis of HIV in individuals at a very early stage of HIVinfection and can be used to prevent HIV-positive blood from beingtransfused into patients.

One advantage of using the above-described method for HIV diagnosis isattributed to the specific response of the recombinant cells to HIVvirus only. Because expression of the reporter gene is specificallyregulated by HIV specific gene products, ambiguity in diagnosis orreport of false positives can be avoided in the clinic. On the otherhand, by using the above-described method, HIV virus may be detected inthose individuals who are infected by HIV but do not have detectablelevels of serum antibody (seronegatives), thereby reducing the incidentsof false negatives which may arise from using antibody-based detectionmethods.

The methods described above can also be used to amplify HIV virus,especially strains with low occurrences in the blood sample and evasiveto other detections. With the replication and amplification of the HIVvirus in the recombinant cells, HIV virus with higher titer can begenerated in the cell culture and isolated for further studies such ascloning of novel HIV strains.

The methods described above can also be used to differentiate strains ortropisms of HIV viruses in a sample by using recombinant cellsselectively expressing certain HIV coreceptors. For example, CXCR4coreceptor which is required by T-tropic strains can be selectivelyexpressed in a first recombinant cell line to allow infection ofT-tropic strains of HIV. Meanwhile, since M-tropic strains require CCR5coreceptor to infect cells, a second recombinant cell line can beconstructed to selectively express CCR5 to allow infection of M-tropicstrains of HIV. By having the first and second recombinant cell linesexpressing different coreceptors, the first and second recombinant celllines can selectively detect T-tropic, M-tropic or dual-tropic strainsin the presence of other strains of HIV virus.

Alternatively, the first recombinant cell line may include a firstreporter gene such as GFP, while the second recombinant cell line mayinclude a second reporter gene such as EBFP. When the first and secondcell lines are mixed in one culture and contacted by a sample containingHIV virus with unknown tropism, selective expression of one reportergene may indicate single tropism of the virus, while expression of bothreporter genes may indicate dual tropism. Different fluorescencesemitted by the first and second cell lines observed under microscope canfacilitate independent identification of each cell line in one culture.

The methods described above can also be used for quantitative analysisof HIV virus in a sample. For example, by using control samples withvarying titers, the viral load can be readily calculated by comparing tothe control samples. Alternatively, the viral titer of a sample can alsobe determined by serially diluting the sample until end point infectionis achieved in multiple cell culture plates, i.e. some of the cellculture plates are infected while the other plates are not infected bythe diluted sample.

3. Methods for Detecting HIV Drug Resistance

Methods are also provided for detecting HIV drug resistance in a sample.These methods may be used to detect whether a course of treatment forHIV infection with one or more drugs is ineffective due to the presenceof one or more strains of HIV which are resistant to the one or moredrugs being used. These methods may also be used to isolate HIV strainswhich are resistant to one or more anti-HIV agents.

In one embodiment, the method comprises:

taking a culture of recombinant cells, which (a) are capable of celldivision, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

contacting the cell culture with a sample containing HIV virus;

adding one or more anti-HIV agents to the cell culture either before orafter contacting the cell culture with the sample; and

detecting a change in a level of expression of the reporter gene in thecells.

Anti-HIV agents used in the methods may be any agents with knownanti-HIV activities, either tested preclinically or clinically. Examplesof anti-HIV agents which may be used to screen for HIV drug resistanceinclude, but are not limited to, nucleoside HIV RT inhibitors such asZIDOVUDINE, DIDANOSINE, ZALCITABINE, LAMIVUDINE, STAVUDINE, ABACAVIR,nonnucleoside RT inhibitors such as NEVIRAPINE, DELAVIRDINE, EFAVIRENZ,protease inhibitors such as INDINAVIR, RITONAVIR, SAQINAVIR, NELFINAVIR,AMPRENAVIR, and combinations thereof.

The culture of recombinant cells used in the method may be any cellwhich has the above described properties. The recombinant cellsdescribed in Section I are examples of cells having these properties andmay be used in this method.

Detecting a change in a level of expression of the reporter gene in thecells in the culture may be performed by detecting a change in a levelof expression of the reporter gene in individual cells or a change in alevel of expression of the reporter gene across the cell culture.

In one embodiment, detecting a change in a level of expression includesdetecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

In another embodiment, detecting a change in a level of expressionincludes comparing a level of expression in cells contacted with thesample to a level of expression cells contacted with one or more controlsamples. For example, cells contacted with a sample containing HIV virusbut not with the one or more anti-HIV agents can serve as a negativecontrol, while cells contacted with a sample containing a HIV virus thatis not known to be resistant to the one or more anti-HIV agents addedmay preferably serve as a positive control. By using suitable controls,induction of the reporter gene expression may be better correlated withthe resistance of the HIV virus to the agents.

It is noted that regulation of the reporter gene may be up regulation ordown regulation. Accordingly, a change in the level of expression of thereporter gene may be an increase or decrease in reporter geneexpression.

In one variation of this embodiment, the cell culture is contacted withone or more anti-HIV drugs before being contacted with a samplecontaining the HIV virus. Alternatively, the cell culture may becontacted with one or more anti-HIV drugs after being contacted with asample containing the HIV virus and incubating for a time sufficient forthe HIV virus replication to occur. This may be particularlyadvantageous for the initial amplification of the HIV virus with lowtiter in the sample before being tested for drug resistance.

The methods described above can be used to detect drug resistance of HIVvirus contained in patient samples, isolated virus stocks orlaboratory-adapted HIV strains. Owing to ultra sensitivity of therecombinant cells to a single HIV virion, the strains of HIV virus thatescape the drug regimen or the ones that are not predominant circulatingvariants can replicate in the cell culture and be isolated for furthergenotypical analysis.

In comparison, the methods that have been used to detect anti-HIV drugresistance are less sensitive, time-consuming and technically demanding.The currently used methods include genotypic assays for detecting HIVgenome mutation based on PCR amplification of the viral RNA followed bysequencing of the amplified DNA templates, and phenotypic assays basedon recombinant HIV virus (Hirsch, M. S. (1998) JAMA 279: 1964-1991).While the most sensitive PCR-based assay that has been developed may notbe sensitive enough to detect plasma HIV RNA below 50 copies/mL, falsepositivity for mutations may be generated due to carry over from otherHIV samples in the laboratory or from random polymerase errors duringPCR. The recombinant virus assay requires a first RT-PCR amplificationof plasma HIV RNA at more than 1000 copies/mL, cloning the viral cDNAinto an HIV vector, and then growing up the virus in permissive cellline. The whole process may take more than two weeks to generate resultsand demand for highly skilled personnel to perform the test.

Thus, the methods provided in the present invention are more sensitivefor detecting replicating HIV virus (at only about 5 virions/mL), moreefficient for testing for HIV drug resistance (less than a week), andmore economic for high throughput screening.

4. Methods for Designing Patient Customized HIV Cocktail Treatments

Methods are also provided for taking a patient known to be infected withone or more strains of the HIV virus and determining what combination ofone or more anti-HIV agents will be effective in treating the patient.These methods can be used when a patient is initially being treated withanti-HIV agents or after a patient has been treated for a period of timewith one or more anti-HIV agents and one or more resistant strains mayhave developed resistance to the anti-HIV agents being used.

In one embodiment, the method comprises:

taking a plurality of cell cultures, each of the cultures containingrecombinant cells (a) are capable of cell division, (b) express CD4receptor and one or more additional cell surface receptors necessary toallow the HIV virus to infect, (c) enable the HIV virus to replicate andinfect the noninfected cells in the culture, and (d) comprises areporter sequence introduced into the recombinant cells comprising areporter gene whose expression is regulated by a protein specific to HIVviruses which is expressed from a genome of an HIV virus upon infectionof the recombinant cell by the HIV virus;

contacting the cell cultures with a sample containing the HIV virus;

adding a different set of one or more anti-HIV agents to each of thecell cultures, either before or after contacting the cell cultures withthe sample; and

comparing expression of the reporter gene in the plurality of cellcultures.

In one variation, each cell culture of the plurality is contacted with adifferent set of one or more anti-HIV agents before being contacted witha sample containing the HIV virus.

In another variation, each cell culture of the plurality is contactedwith a different set of one or more anti-HIV drugs after being contactedwith a sample containing the HIV virus and incubating for a timesufficient for the HIV virus replication to occur.

The anti-HIV agents can be any agents with known anti-HIV activities,such as the ones described in Section 3, and combinations thereof.

The culture of recombinant cells used in the method may be any cellwhich has the above described properties. The recombinant cellsdescribed in Section I are examples of cell having these properties andmay be used in this method.

Detecting a change in a level of expression of the reporter gene in thecells in the culture may be performed by detecting a change in a levelof expression of the reporter gene in individual cells or a change in alevel of expression of the reporter gene across the cell culture.

In one embodiment, detecting a change in a level of expression includesdetecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

In yet another variation of this embodiment, the method further includescomparing the change in the level of expression of the reporter genewhen different or no anti-HIV agents are used. For example, arecombinant cell culture that is contacted with the sample containingthe HIV virus but not with the one or more anti-HIV agents can serve asa negative control, while a recombinant cell culture that is contactedwith a sample containing HIV virus or a modified adenovirus, and the oneor more anti-HIV agents can serve as a positive control. By usingsuitable controls, inhibition of the reporter gene expression may bebetter correlated with anti-HIV efficacy of the agents.

It is noted that regulation of the reporter gene may be up regulation ordown regulation. Accordingly, a change in the level of expression of thereporter gene may be an increase or decrease in reporter geneexpression.

In one variation of this embodiment, the cell culture is contacted withone or more anti-HIV agents before being contacted with a samplecontaining the HIV virus. Alternatively, the cell culture may becontacted with one or more anti-HIV agents after being contacted with asample containing the HIV virus and incubating for a time sufficient forthe HIV virus replication to occur. Such preamplification of the HIVvirus may be advantageous for patient samples containing lower titer ofthe HIV virus to be tested against the anti-HIV agents.

The methods provided in this section can be used for screening ananti-HIV agent or agent combinations that are most active in inhibitingHIV viral infection and/or replication. The screening can be conductedagainst virtually all strains of HIV virus, regardless of theirgenotypes or tropisms. The results generated can help the physician ofHIV infected patients monitor HIV drug resistance, optimize the drugregimen and use the most efficacious drug “cocktail” to treat thepatient. By using such drug cocktails customized for each individualpatient and adjusted during the course of the treatment, physicians maysuccessfully prevent the HIV virus from developing drug resistance.Furthermore, physicians can avoid unnecessary side effects and drugtoxicity that would otherwise arise from treating a patient withineffective anti-HIV agents.

The ample and stable supply of the recombinant cells used in thesemethods, as well as the ease of culturing the cells, enables one to usethe methods provided in this section in a high throughput screeningformat to test many more drug cocktail combinations than would otherwisehave been possible. Furthermore, because the HIV virus contained in thesample from a patient may potentially harbor drug resistances strains,conventional drug screening may not have been effective in finding theoptimum drug regimen. By using the methods provided in this section, themost efficacious drug regimen may be readily identified by designing andtesting exhaustive combinations of different drugs that target differentcomponents of the HIV virus or HIV receptors.

5. Methods for Screening Compositions for Anti-HIV Activity

The present invention also relates to methods for screening compositionswhich are not known to have anti-HIV activity for anti-HIV activity. Asused herein, a composition is intended to refer to any composition ofmatter, including single molecules, macromolecules such as proteins andnucleotides, or combinations of two or more molecules or macromolecules.

In one embodiment, the method comprises:

taking a culture of recombinant cells, which (a) are capable of celldivision, (b) express CD4 receptor and one or more additional cellsurface receptors necessary to allow the HIV virus to infect, (c) enablethe HIV virus to replicate and infect the noninfected cells in theculture, and (d) comprise a reporter sequence introduced into therecombinant cells comprising a reporter gene whose expression isregulated by a protein specific to HIV viruses which is expressed from agenome of an HIV virus upon infection of the recombinant cell by the HIVvirus;

contacting the cell culture with a sample containing the HIV virus;

adding one or more agents whose anti-HIV activity are unknown to thecell culture either before or after contacting the cell cultures withthe sample; and

detecting a change in a level of expression of the reporter gene in thecells in the culture.

The culture of recombinant cells used in the method may be any cellculture which has the above described properties. The recombinant cellsdescribed in Section I are examples of cells having these properties andmay be used in this method.

The agents can be any anti-HIV drug candidates from natural sources orsynthetically generated. The agents can be any agent targeting anycomponents of the HIV virus, such as RT inhibitors, protease inhibitors,antisense and ribozyme oligonucleotides against HIV mRNA or viral RNAgenome, decoys of TAR sequence or RRE (rev response element),competitive inhibitors like soluble CD4, Gag or Env protein mutants, andagents that bind to HIV receptor or coreceptors and block the entry ofHIV into the host cells.

Detecting a change in a level of expression of the reporter gene in thecells in the culture may be performed by detecting a change in a levelof expression of the reporter gene in individual cells or a change in alevel of expression of the reporter gene across the cell culture.

In one embodiment, detecting a change in a level of expression includesdetecting whether viral replication within the cell culture hasoccurred. Viral replication may be detected by detecting which cells areinitially infected, and detecting a change in a level of expression ofcells in the cell culture which were not initially infected.

In another embodiment, detecting a change in a level of expressionincludes comparing a level of expression in a sample to a level ofexpression in one or more control samples. For example, a recombinantcell culture that is contacted with a sample containing HIV virus butnot with any potentially anti-HIV agents can serve as a negativecontrol, while a recombinant cell culture that is contacted with asample containing an HIV virus and the one or more agents that are knownto have anti-HIV activity can serve as a positive control. By usingsuitable controls, regulation of the reporter gene expression may bebetter correlated with anti-HIV efficacy of the agents.

It is noted that regulation of the reporter gene may be up regulation ordown regulation. Accordingly, a change in the level of expression of thereporter gene may be an increase or decrease in reporter geneexpression.

In one variation of this embodiment, the cell culture is contacted withone or more agents before being contacted with a sample containing theHIV virus. Alternatively, the cell culture may be contacted with one ormore agents after being contacted with a sample containing the HIV virusand incubating for a time sufficient for the HIV virus replication tooccur. This may be particularly advantageous for the initialamplification of the HIV virus with low titer in the sample before beingtested against the agents.

The methods described above can be used for high throughput screeningfor anti-HIV drug candidates against various HIV containing samples,especially for libraries of compounds generated by combinatorialchemistry. These methods may be performed in any format that allowsrapid preparation and processing of cells contained in multiple-wellplates, such as 96-well plates. Stock solutions of the test agent aswell as other assay reagents may be prepared manually and all subsequentpipetting, diluting, mixing, washing, incubating, sample readout anddata collecting may be done using commercially available roboticpipetting equipment, automated work stations, analytical instruments fordetecting the signal generated by the assay. Examples of such detectorsinclude, but are not limited to, spectrophotometers, calorimeters,luminometers, fluorometers, and devices that measure the decay ofradioisotopes.

The methods described above are particularly cost-effective for use inhigh throughput screening because the recombinant cells areimmortalized, easy to culture and more stable, compared to primary humancells such as PBMC cells. Furthermore, effects of multiple agents atmultiple doses on HIV infection and replication can be directlymonitored by detecting levels of reporter gene products in the 96-cellculture plates on a calorimetric or fluorescence plate reader.

6. Constructing a Recombinant Cell Line According to the PresentInvention

The recombinant cells used in the present invention are immortalizedcells. Human tumor cell lines are preferably used. Other transformednormal cells, such as human transformed primary embryonal kidney 293cells, and human primary cells immortalized by transfection withtelomerase (Bodnar, A. G. et al. (1998) Science 279:349-352) can also beused.

In order to create a cell line which is permissive to HIV infection, CD4and one or more other HIV receptors are transfected, transduced orotherwise introduced into the immortalized cells. The one or more otherHIV receptors preferably include CXCR4 and CCR5 receptors.

CD4 receptor is believed to be the primary receptor for HIV entry intothe host cell. It has recently been discovered that specific chemokinereceptors such as CXCR4 and CCR5 receptors play important roles inmediating HIV entry and tropism for different target cells (reviewed byBerger, E. a. (1997) AIDS 11; Suppl. a: S3-S16; Dimitrov, D. S. (1997)Cell 91: 721-730). Macrophages-tropic (M-tropic) strains of HIV viruscan replicate in primary CD4⁺ T cells and macrophages and use thebeta-chemokine receptor CCR5 and less often, CCR3 receptor. T cellline-tropic (T-tropic) HIV strains can also replicate in primary CD4⁺ Tcells but can in addition infect established CD4⁺ T cell lines in vitrovia the alpha-chemokine receptor CXCR4. Many of the T-tropic strains canuse CCR5 in addition to CXCR4. Chemokine receptor-like HIV coreceptorSTRL33 is expressed in activated peripheral blood lymphocytes and T-celllines and can function as an entry cofactor for Env proteins fromM-tropic, T-tropic and dual tropic strains of HIV-1 and SIV. Other HIVcoreceptors have also been identified by numerous in vitro assays,including chemokine receptors CCR2b, CCR3, CCR8 and CX3CR1 as well asseveral chemokine receptor-like orphan receptor proteins such asGPR15/BOB and STRL33/BONZO. Each or a set of these HIV coreceptors canmediate entry of different strains of HIV virus into the host cell. Bytransfecting, transducing or otherwise introducing these receptors intothe immortalized cell line, the host cell line can be renderedpermissive to HIV strains with broad-spectrum tropisms. In particular,by cell-cell fusion of the immortalized cell with cells expressing cellsurface receptors known to be involved in HIV infection such as T-cellsor monocytes, the immortalized cell can be transduced with various HIVreceptors simultaneously.

By transfecting, transducing or otherwise introducing a selected set ofcoreceptors into an immortalized cell line or selectively expressingcertain coreceptors on the cell surface, a cell line can be designedwhich is permissive to certain strains of HIV and is not be permissiveto other strains of HIV. For example, CXCR4 coreceptor which is requiredby T-tropic strains can be selectively expressed in the recombinantcells to allow infection of T-tropic strains of HIV. Meanwhile, M-tropicstrains require CCR5 coreceptor to infect cells. By having therecombinant cells not express CCR5 coreceptor, the recombinant cell linecan selectively detect T-tropic strains in the presence of M-tropicstrains.

In order to detect HIV infection with a high level of sensitivity, a“molecular switch” with high induction ratio is introduced into theimmortalized cell line expressing CD4 receptor and the one or moreadditional HIV receptors. The molecular switch comprises a reporter genewhose expression is induced when the cells are infected by HIV. Variousreporter genes can be used including lacZ (encoding β-galactosidase),luciferases gene, CAT gene, SEAP gene, and genes encoding fluorescentproteins such as green fluorescent protein (GFP), enhanced bluefluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP)and enhanced cyan fluorescent protein (ECFP).

The promoter region for the reporter gene contains a basic promoter anda single or multiple copies of HIV specific enhancer sequence. The basicpromoter can be any cellular or viral basic promoters such as the basicpromoter regions of β-actin promoter, insulin promoter, humancytomegalovirus (CMV) promoter, HIV-LTR (HIV-long terminal repeat), Roussarcoma virus RSV-LTR, and simian virus SV40 promoter. The HIV specificenhancer sequence can be any sequence that can regulate the expressionof the reporter gene via direct or indirect interaction with one or moreHIV specific gene products. For example, the responsive element (TAR)for HIV transactivator protein Tat can be used to enhance the expressionof the reporter gene. Upon infection of HIV, Tat expressed from theviral genome binds to TAR sequence and, coupled with the basic promoter,induces expression of the reporter gene. More than one copy of TARsequence can be linked to further enhance expression of the reportergene and raise the induction ratios.

Alternatively, expression of the reporter gene can be induced byprotein-protein interactions between an HIV gene product, a DNA-bindingprotein (e.g. GAL4 DNA binding domain). a transactivator protein (e.g.VP16 transactivator domain derived from herpes simplex virus) that areexpressed by the host cell. Upon binding of the HIV specific geneproduct to the DNA binding protein as well as to the transactivatorprotein, reconstitution of a transcription factor is achieved bybringing the DNA-binding protein and the transactivator protein intoclose approximately. The reconstituted transcription factor can thenactivate downstream reporter gene expression via the specific bindingbetween the enhancer sequence (e.g. GAL4 enhancer sequence) upstream ofthe basic promoter with the DNA binding protein.

It should be noted that expression of a reporter gene can also beindirectly regulated by an HIV specific protein. For example,transcription of the reporter gene can be under the control a strongpromoter, such as the bacteriophage T7 or SP6 promoters, whileexpression of T7 or SP6 polymerase is regulated by a promoter comprisinga basic promoter and an HIV specific enhancer sequence. Upon binding ofthe HIV specific protein to the enhancer sequence, expression of T7 orSP6 polymerase is enhanced. As a result, T7 or SP6 polymerase expressedin the cell can then bind to the T7 or SP6 promoter upstream of thereporter gene and induce expression of the reporter gene in the cell.

Various methods can be used to introduce genes into the immortalizedcells. Examples of methods that may be used include, but are not limitedto, calcium phosphate-mediated direction transfection, liposome-assistedtransfection, and virus-mediated transfection. HIV receptors can also beintroduced into the host cell through cell fusion with natural cellsexpressing these receptors on the cell surface. Clones of cellsexpressing the transfected genes may be selected by antibiotics such ashygromyin, G418, zeocin, etc., or based on herpes simplex virus tk gene.Expression of each receptor gene may be confirmed by Western blot todetect the protein with an antibody, Northern blot to detect the RNAwith a nucleotide probe, or by FACS using the HIV receptor expressed onthe cell surface as antigens.

Two examples of plasmid vectors containing HIV receptor genes and areporter gene are diagramed in FIGS. 1A and 1B.

As illustrated in FIG. 1A, CD4 and HIV co-receptors are expressed fromSV40 early and late promoters in opposite directions. Genes encoding CD4and CCR5 receptors are expressed from SV40 early promoter by a splicingmechanism at the SA sites. Genes encoding CXCR4 and hygromycinresistance are expressed bicistronically from SV40 late promoter withHygro being separated by an internal ribosome entry site (IRES).Expression of hygromycin resistance gene enables selection of the cell.The plasmid also contains prokaryotic replication origin andampicillin-resistance gene for DNA propagation in bacteria. The reportergene is carried by a separate plasmid that contains a second selectiongene (tk). The two plasmids may be co-transfected into HeLa cellssimultaneously or sequentially. Cell clones expressing all of thetransfected genes can be selected with antibiotics accordingly.

Genes encoding HIV receptor and coreceptors may also be expressed fromthe two retroviral vectors illustrated in FIG. 1B. The receptors geneare expressed from the murine leukemia virus (MLV) LTR-promoter, eachprotein is expressed from a spliced mRNA or from an IRES (B.1). Thereporter sequence is carried by a second retroviral vector.Transcription of the reporter gene is in the opposite direction of theMLV LTR promoter with the enhancer sequence deleted in order to preventunregulated expression from the LTR promoter (B.2).

These vectors are packaged into infectious but replication-incompetentvirions by using a packaging cell line, such as those stable ortransient production lines based on the 293T cell line. The packagingcell line expresses all the necessary proteins, Gag, Pol and Env, thatare required for packaging, processing, reverse transcription, andintegration of recombinant retroviral genome containing the Psipackaging signal.

The retroviral vectors are transfected into the packaging cell line. Thevirions produced in the packaging cells are then collected and used toinfect a target cell. Since the virons are replication-incompetent, thegenes carried by the retroviral vectors are stably integrated into thetarget cell genome and can be expressed under the control of theupstream promoter without producing infectious virions. The cellsexpressing all of the transduced genes can be selected with antibioticsand confirmed by Northern, Western blots or FACS accordingly.Alternatively, the cells expressing the reporter sequence can beselected by infecting the cell culture with a modified adenoviruscarrying HIV specific gene such as tat.

It should be noted that expression of HIV receptors can also becontrolled by an inducible promoter such as a tetracycline responsiveelement TRE. For example, one or more of the HIV coreceptors can beselectively presented on the cell surface by a controlled expressionusing the Tet-on and Tet-off expression systems provided by Clontech(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). In the Tet-on system, gene expression is activated by theaddition of a tetracycline derivative doxycycline (Dox), whereas in theTet-off system, gene expression is turned on by the withdrawn oftetracyline (Tc) or Dox. Any other inducible mammalian gene expressionsystems may also be used. Examples include systems using heat shockfactors, steroid hormones, heavy metal ions, phorbol ester andinterferons to conditionally expressing genes in mammalian cells.

Overall, the present invention provides novel recombinant cell lines andmethods using these cell lines. These methods are convenient,cost-effective and ultra sensitive for the detection of HIV infectionand replication. These methods can be very useful for high throughputscreening in preclinical drug discovery and development, as well asdesigning more efficacious anti-HIV drug cocktails in the clinic tocombat HIV drug resistance.

EXAMPLE

1. Productive Infection of Recombinant HeLa Cells with HIV Virus

A recombinant cell line was established from human cervical cancer HeLacells. The HeLa cells were cotransfected with an expression vector(pRepD4R4) and a vector (pTAR3Clac) at a 1:1 ratio. As shown in FIG. 2Athe expression vector pRepD4R4 includes CD4 receptor and CXCR4 receptorgenes that are separated by an IRES sequence. As shown FIG. 2B thevector pTAR3Clac includes a reporter sequence comprising a promoterregion that includes three copies of TAR sequences and a CMV basicpromoter, and a lacZ reporter gene whose expression is under the controlof the promoter. The stably-transfected cells were selected by culturingin medium containing G418 at 900 μg/ml. Each clone of the cells selectedwas subsequently cultured in duplicates, and one of the duplicates wasinfected with a low titer HIV stock solution. The low-titer HIV stockwas collected from supernatant of a HeLa cell culture that wastransfected with a B-cell tropic HIV provirus DNA (strain GRCSF) andincubated for 3 days post transfection.

Upon infection of HIV contained in the stock solution, Tat proteinexpressed from the viral genome binds to TAR and induces expression oflacZ reporter gene to produce high level of β-galactosidase. The cellclones expressing β-galactosidase and stained blue with X-gal wereidentified, and the cells from the uninfected duplicate of the darkestblue colony were propagated. Such selected cells were designated asHeLaD4R4 cells.

HeLaD4R4 cells constructed as described above were tested for HIVinfection. HeLaT4 cells (also called HT4) which express human CD4receptor were used as a control. The HeLaD4R4 cells and HeLaT4 cellswere grown up in DMEM and 5% bovine calf serum.

Exponentially growing cells were cultured in a six-well plate andinfected with 1 ml of a diluted HIV stock (about 10 infectiousparticles/ml) obtained from HIV provirus transfected HeLa cell cultureas described above. The cells were continuously cultured, and fixed with1% formaldehyde for 2 minutes 1, 3, 4, 5 days after the initialinfection. The cells were fixed with 0.5% formaldehyde for 2 min. andstained with X-gal (0.5%) at 37° C. over night. Since the lacZ reportergene product, β-galactosidase, converts the substrate from colorless todark blue, cells expressing β-galactosidase as a result of beinginfected with HIV appear distinctly blue.

FIG. 3A shows the control HeLaT4 cells after three days of being exposedto HIV. As can be seen, almost all of the HeLa cells were not stainedblue, with few cell stained faint blue. This indicates that cellswithout HIV CXCR4 were poorly infected and the HIV virus did notreplicate within the cell culture.

FIGS. 3B-2E shows HeLaD4R4 after 1, 3, 4, and 5 days. As can be seen inFIG. 3B, infection can be readily detected after 1 day, as shown by theblue cells. As can be seen in FIGS. 3C and 3D respectively,progressively more cells were infected and stained blue after 3 and 4days. As can be seen in FIG. 3E, virtually all cells in the well wereinfected and stained dark blue after 5 days.

The results shown in FIGS. 3B-3E indicate that following initialinfection of a few cells by about ten HIV virions, HIV was able toundergo a productive infection, i.e. an infection of a cell which isfully permissive for virus replication and production of progeny virions(Stevenson, M. AIDS 11 Suppl. a: S25-S33). In addition, the infectedcells appear to retain normal morphology, i.e. remaining attached to thesubstrate of the culture plate instead of rounding up and detaching fromthe plate

The results shown in FIG. 3E are particularly significant because HIVvirions initially added to the sample were able to replicate within thecell culture and spread to infect other cells that are not infectedoriginally (compare FIGS. 3B and 3E). This is in significant contrast toan increase of cells stained blue simply due to cell division.

FIG. 3F illustrates a further experiment where AZT (100 μg/ml) was addedto inhibit HIV replication and infection. As can be seen in FIG. 3F,after four days of incubation in the presence of AZT only a few clustersof cells were infected and stained blue. The sparse clusters of bluecells are most likely cells divided from the few cells that wereinitially infected by the HIV virions added to the well.

By comparing FIG. 2F to FIGS. 3B-3E, one can see that AZT was effectiveas an anti-HIV agent since the expression of the reporter gene wassignificantly reduced due to the presence of AZT. This comparison of theresults in FIG. 3F to FIGS. 3B-3E is an example of how the presentinvention can be used to detect HIV drug resistance and to screencompositions for anti-HIV activity.

2. Method for HIV Diagnosis

An example is provided for detecting HIV in a sample. This method can beused to diagnose a patient infected with HIV. According to the method,recombinant cells are seeded into a multiple well plate. a small amountof serum from an individual to be tested is added to duplicates of thewells. After two to four days incubation, the cells are processed andthe results are analyzed depending on the type of reporter gene used.For example, when lacZ gene is used as the reporter gene for therecombinant cells, the cells are treated with a processing solutioncontaining the substrate X-Gal for β-galactosidase, low concentration offormaldehyde (1%) and glutaraldehyde the (0.1%) to gently fix the cellswhile not inactivating the reporter protein. When a green fluorescentprotein (GFP) gene is as the reporter gene for the recombinant cells,the cells are observed under an UV microscope directly. The presence ofcells emitting green fluorescence indicate that the cells may have beeninfected by HIV virus contained in the sample. By using GFP as areporter gene replication of HIV can be directed monitored any timeduring the incubation without fixing and processing cells to ensure thatenough HIV virus has been replicated within the culture.

The above-described diagnosis test can be used as an independent testfor HIV infected patients, or in conjunction with HIV drug resistanceand other HIV diagnosis tests.

a positive control agent may be used to ensure that the recombinantcells are responsive to HIV infection. a defective common cold virusstrain carrying an HIV tat gene that encodes HIV transactivator proteinTat may be used as a positive control agent. The common cold virus isused as a vector to transfer the HIV tat gene into cells to mimic HIVinfection. HIV itself may not be ideal for use as a positive controlbecause HIV may not be sufficient stable and can easily lose itsactivity, thus the virus may not be stored for an extended period oftime. In contrast, the common cold virus can be dried into powder andstored for a long time. In addition, this strains of common cold virusis derived from a strain of common cold virus (adenovirus type 5) thatis defective in viral replication, therefore safer for an extensiveusage as a positive control.

3. Method for Detecting HIV Drug Resistance

An example of how to perform the method for detecting HIV drugresistance is provided. Recombinant cells are seeded into each well of amultiple-well plate. Duplicate wells contain each anti-HIV agent to betested. a small amount of patient serum is added to each well andincubated for a few days. After two to four days of incubation, thecells are processed and the results are analyzed depending on the typeof reporter gene used. For example, when lacZ gene is used as thereporter gene for the recombinant cells, the cells are treated withprocessing solution containing the substrate X-Gal for β-galactosidase,low concentration of formaldehyde (1%) and glutaraldehyde the (0.1%) togently fix the cells while not inactivating the reporter protein. Forquantitative analysis, levels of beta-Gal can be measured by an ONPGassay on the cell extract. When a green fluorescent protein (GFP) geneis used as the reporter gene for the recombinant cells, the cells areobserved under an UV microscope directly. For quantitative analysis, thefluorescent cells are sorted by FACS and numbers of cells expressing theGFP reporter are measured.

If the cells in the wells containing a particular drug express thereporter gene at a sufficient level, it indicates that the HIV viruscontained in the sample may be resistant to the drug at the tested dose,and the virus has replicated and spread the infection among therecombinant cells in the presence of the anti-HIV drug.

Wells where no serum sample has been added can be used as a negativecontrol. Negative controls can be performed for each agent being tested.a positive control, for example using the positive control agentdescribed in Example 2 (adenovirus carrying HIV tat gene), can also beperformed for each agent tested to ensure that the recombinant cellsfunction properly.

4. Method for Determining Viral Load in a Patient Serum

An example of how to perform the method for determining viral load inpatient serum is provided. About 1 milliliter of patient's serum isdiluted progressively, such as 1:10, 1:100, 1:1000, etc, and added towells containing the recombinant cells. The highest dilution that stillinduces expression of the reporter gene of the recombinant cells in thewell is the titer (concentration) of the HIV in the patient serum. Whenthe viral load become low, finer steps of dilution may be performed todetermine more accurately the numbers of viral particles in thepatient's serum.

The method can be used to determine how many viral particles permilliliter are present in patient serum. Since the recombinant cells ina culture are sensitive to infection of even a single virion, thismethod can detect infection by only one viral particle, thereforesuitable for detecting a patient sample containing low titer HIV, even afew viral particles per milliliter of patient serum. Such a highsensitivity is important for monitoring the progress of anti-HIV drugtreatment. Compared to the “ultra-sensitive” PCR-based assays that canonly detect hundreds or more viral particles per milliliter of patientserum, this method is more sensitive and can be used to detect muchlower titer HIV in the sample. This is particularly important fordetecting HIV virus in a patient sample after anti-HIV drug treatmentwhen viral titer is below the detectable level of conventional HIVdetection methods.

5. Method for Screening for Anti-HIV Agents

Described here is an example of a method for performing high throughputanti-HIV drug screening. To screen for new anti-HIV agents, therecombinant cells are seeded into a multiple well plate, such as 96-wellplate. To each well the agent to be tested for anti-HIV activity isadded. a small amount of HIV stock is added to each well, so that thecells in each well are infected with about 10 viral particles. After afew days of incubation, the cells in the wells are analyzed on acalorimetric or fluorescence plate reader. The wells are compared withone or more wells containing the recombinant cell and virus but not theagent. Inhibition of the expression of the reporter gene in wellscontaining an agent indicates that the agent may have anti-HIV activityat the tested dose. Once potential anti-HIV agents have been identified,the test may be repeated to further confirm the anti-HIV activity of theagent.

Throughout this application, various publications are referenced. Thedisclosures of these publications, and the references cited therein, intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexample be considered as exemplary only, with a true scope and spirit ofthe invention being indicated by the claims.

1. A method for detecting HIV drug resistance in a sample, comprising:taking a culture of recombinant cells in which at least one of therecombinant cells comprises (a) a reporter sequence comprising areporter gene whose expression is regulated by a protein specific toHIV, and (b) a heterologous sequence which encodes CD4 and one or moreadditional cell surface receptors, wherein the heterologous sequenceexpresses CD4 and the one or more additional cell surface receptors atelevated levels as compared to the cell in the absence of expression bythe heterologous sequence such that productive infection of therecombinant cell by the HIV is achieved, which is defined by HIV viralreplication and the infection of non-infected cells in the culture ofthe recombinant cells; contacting the cell culture with a samplecontaining HIV; adding one or more anti-HIV agents to the cell cultureeither before or after contacting the cell culture with the sample; anddetecting a change in a level of expression of the reporter gene in thecells.
 2. The method according to claim 1, wherein a different set ofone or more anti-HIV agents is added to each cell culture of a pluralityof the culture of recombinant cells before the culture of recombinantcells is contacted with a sample containing the HIV virus.
 3. The methodaccording to claim 1, wherein a different set of one or more anti-HIVagents is added to each cell culture of a plurality of the culture ofrecombinant cells after the culture of recombinant cells is contactedwith a sample containing the HIV virus.
 4. The method according to claim1, wherein the anti-HIV agents include agents selected from the groupconsisting of nucleoside HIV RT inhibitors, nonnucleoside RT inhibitors,and protease inhibitors.
 5. The method according to claim 1, wherein theanti-HIV agents include agents selected from the group consisting ofZIDOVUDINE, DIDANOSINE, ZALCITABINE, LAMIVUDINE, STAVUDINE, ABACAVIR,NEVIRAPINE, DELAVIRDINE, EFAVIRENZ, INDINAVIR, RITONAVIR, SAQINAVIR,NELFINAVIR, and AMPRENAVIR.
 6. A method for screening compositions foranti-HIV activity, comprising: taking a culture of recombinant cells inwhich at least one of the recombinant cells comprises (a) a reportersequence comprising a reporter gene whose expression is regulated by aprotein specific to HIV, and (b) a heterologous sequence which encodesCD4 and one or more additional cell surface receptors, wherein theheterologous sequence expresses CD4 and the one or more additional cellsurface receptors at elevated levels as compared to the cell in theabsence of expression by the heterologous sequence such that productiveinfection of the recombinant cell by the HIV is achieved, which isdefined by HIV viral replication and the infection of non-infected cellsin the culture of the recombinant cells; contacting the cell culturewith the HIV; adding one or more agents whose anti-HIV activities areunknown prior to the screening to the cell culture either before orafter contacting the cell cultures with the HIV virus; and detecting achange in a level of expression of the reporter gene in the cells in theculture.
 7. The method according to claim 6, wherein the agents areadded to the cell culture before the cell culture is contacted with theHIV virus.
 8. The method according to claim 6, wherein the agents areadded to the cell culture after the cell culture is contacted with theHIV virus.
 9. A method for detecting HIV drug resistance in a sample,comprising: taking a culture of recombinant cells wherein at least oneof the recombinant cells comprises a reporter sequence comprising areporter gene whose expression is regulated by a protein specific toHIV, and the recombinant cell is genetically modified to express CD4 andone or more additional cell surface receptors at elevated levelsrelative to a non-genetically modified form of the recombinant cell suchthat productive infection of the recombinant cells by the HIV virus isachieved, which is defined by HIV viral replication and the infection ofnon-infected cells in a culture of the recombinant cell; contacting thecell culture with a sample containing HIV; adding one or more anti-HIVagents to the cell culture either before or after contacting the cellculture with the sample; and detecting a change in a level of expressionof the reporter gene in the cells.
 10. The method according to claim 9,wherein the reporter gene expression is up-regulated by the HIV specificprotein.
 11. The method according to claim 9, wherein the HIV specificprotein is an HIV transactivator protein.
 12. The method according toclaim 9, wherein the HIV transactivator protein is Tat.
 13. The methodaccording to claim 9, wherein the reporter sequence comprises apromoter, an HIV virus specific enhancer sequence, and a reporter genewhose expression is regulated by binding of an HIV specifictransactivator protein to the HIV specific enhancer sequence.
 14. Themethod according to claim 13, wherein the HIV specific transactivatorprotein is Tat and the HiM specific enhancer sequence comprises at leastone copy of TAR sequence.
 15. The method according to claim 13, whereinthe HIV specific transactivator protein is Tat and the HIV specificenhancer sequence comprises at least two copies of TAR sequence.
 16. Themethod according to claim 9, wherein the reporter gene is selected fromthe group consisting of genes encoding β-galactosidase, luciferase,β-glucuronidase, chloramphenicol acetyl transferase (CAT), secretedembryonic alkaline phosphatase (SEAP), hormones and cytokines.
 17. Themethod according to claim 9, wherein the one or more additional cellsurface receptors expressed by the recombinant cell are selected fromthe group consisting of CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8,CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO and GPR15/BOB.
 18. The methodaccording to claim 9, wherein the one or more additional cell surfacereceptors expressed by the recombinant cell comprises CXCR4.
 19. Themethod according to claim 9, wherein the one or more additional cellsurface receptors expressed by the recombinant cell comprises CCR5. 20.The method according to claim 9, wherein the one or more additional cellsurface receptors expressed by the recombinant cell comprises CXCR4 andCCR5.
 21. The method according to claim 9, wherein the recombinant cellexpresses a sufficient number of cell surface receptors to render therecombinant cell permissive to substantially all strains of HIV.
 22. Themethod according to claim 9, wherein the recombinant cell is a tumorcell.
 23. The method according to claim 9, wherein the recombinant cellis a cell which has been immortalized by introducing a gene into thecell which renders the cell line immortalized.
 24. The method accordingto claim 9, wherein the recombinant cell is capable of achievingproductive infection of a clinically isolated HIV.
 25. The methodaccording to claim 9, wherein the CD4 receptor and the one or moreadditional cell surface receptors are separately expressed by two ormore retroviral vectors transduced into the recombinant cell.
 26. Themethod according to claim 9, wherein the CD4 receptor and the one ormore additional cell surface receptors are expressed by an adenoviralvector transduced into the recombinant cell.
 27. The method according toclaim 9, wherein the sample containing HIV is a clinical isolate of HIV.28. The method according to claim 9, wherein the sample containing HIVis blood isolated from an HIV-infected individual.
 29. The methodaccording to claim 9, wherein the sample containing HIV is plasmaisolated from an HIV-infected individual.